The epigenetic enzyme DOT1L orchestrates vascular smooth muscle cell-monocyte crosstalk and protects against atherosclerosis via the NF-κB pathway.

AIMS: Histone H3 dimethylation at lysine 79 is a key epigenetic mark uniquely induced by methyltransferase disruptor of telomeric silencing 1-like (DOT1L). We aimed to determine whether DOT1L modulates vascular smooth muscle cell (VSMC) phenotype and how it might affect atherosclerosis in vitro and in vivo, unravelling the related mechanism. METHODS AND RESULTS: Gene expression screening of VSMCs stimulated with the BB isoform of platelet-derived growth factor led us to identify Dot1l as an early up-regulated epigenetic factor. Mouse and human atherosclerotic lesions were assessed for Dot1l expression, which resulted specifically localized in the VSMC compartment. The relevance of Dot1l to atherosclerosis pathogenesis was assessed through deletion of its gene in the VSMCs via an inducible, tissue-specific knock-out mouse model crossed with the ApoE-/- high-fat diet model of atherosclerosis. We found that the inactivation of Dot1l significantly reduced the progression of the disease. By combining RNA- and H3K79me2-chromatin immunoprecipitation-sequencing, we found that DOT1L and its induced H3K79me2 mark directly regulate the transcription of Nf-κB-1 and -2, master modulators of inflammation, which in turn induce the expression of CCL5 and CXCL10, cytokines fundamentally involved in atherosclerosis development. Finally, a correlation between coronary artery disease and genetic variations in the DOT1L gene was found because specific polymorphisms are associated with increased mRNA expression. CONCLUSION: DOT1L plays a key role in the epigenetic control of VSMC gene expression, leading to atherosclerosis development. Results identify DOT1L as a potential therapeutic target for vascular diseases.

miR-128-3p Is a Novel Regulator of Vascular Smooth Muscle Cell Phenotypic Switch and Vascular Diseases.

RATIONALE: MicroRNAs (miRNAs, miRs) are small noncoding RNAs that modulate gene expression by negatively regulating translation of target genes. Although the role of several miRNAs in vascular smooth muscle cells (VSMCs) has been extensively characterized, the function of miRNA-128-3p (miR-128) is still unknown. OBJECTIVE: To determine if miR-128 modulates VSMC phenotype and to define the underlying mechanisms. METHODS AND RESULTS: We screened for miRNAs whose expression is modulated by an altered DNA methylation status in VSMCs, and among the hits, we selected miR-128. We found that miR-128 was expressed in various tissues, primary murine cells, and pathological murine and human vascular specimens. Through gain- and loss-of-function approaches, we determined that miR-128 affects VSMC proliferation, migration, differentiation, and contractility. The alterations of those properties were dependent upon epigenetic regulation of key VSMC differentiation genes; notably, Kruppel-like factor 4 was found to be a direct target of miR-128 and able to modulate the methylation status of the pivotal VSMC gene myosin heavy chain 11 (Myh11). Finally, in vivo lentiviral delivery of miR-128 prevented intimal hyperplasia in a mouse model of carotid restenosis without modifying vital cardiovascular parameters. CONCLUSION: miR-128 is a critical modulator of VSMCs and is regulated by epigenetic modifications upon stress. Its modulation in the context of disease could be exploited for therapeutic purposes.

Circ_Lrp6, a Circular RNA Enriched in Vascular Smooth Muscle Cells, Acts as a Sponge Regulating miRNA-145 Function.

RATIONALE: microRNAs (miRNAs) modulate gene expression by repressing translation of targeted genes. Previous work has established a role for miRNAs in regulating vascular smooth muscle cell (VSMC) activity. Whether circular RNAs are involved in the modulation of miRNA activity in VSMCs is unknown. OBJECTIVE: We aimed to identify circular RNAs interacting with miRNAs enriched in VSMCs and modulating the cells' activity. METHODS AND RESULTS: RNA sequencing and bioinformatics identified several circular RNAs enriched in VSMCs; however, only one, possessing multiple putative binding sites for miR-145, was highly conserved between mouse and man. This circular RNA gemmed from alternative splicing of Lrp6 (lipoprotein receptor 6), a gene highly expressed in vessels and implicated in vascular pathologies and was thus named circ_Lrp6. Its role as a miR-145 sponge was confirmed by determining reciprocal interaction through RNA immunoprecipitation, stimulated emission depletion microscopy, and competitive luciferase assays; functional inhibition of miR-145 was assessed by measuring expression of the target genes ITGß8 (integrin-ß8), FASCIN (fascin actin-bundling protein 1), KLF4 (Kruppel-like factor 4), Yes1 (YES proto-oncogene 1), and Lox (lysyl oxidase). The interaction was preferentially localized to P-bodies, sites of mRNA degradation. Using loss- and gain-of-function approaches, we found that circ_Lrp6 hindered miR-145-mediated regulation of VSMC migration, proliferation, and differentiation. Differential expression of miR-145 and circ_Lrp6 in murine and human vascular diseases suggests that the ratio of circ_Lrp6 bound to miR-145 versus unbound could play a role in vascular pathogenesis. Viral delivery of circ_Lrp6 shRNA prevented intimal hyperplasia in mouse carotids. CONCLUSIONS: circ_Lrp6 is an intracellular modulator and a natural sponge for miR-145, counterbalancing the functions of the miRNA in VSMCs.

Epigenetics and vascular diseases.

Cardiovascular disease remains the number one cause of death and disability worldwide despite significant improvements in diagnosis, prevention, and early intervention efforts. There is an urgent need for improved understanding of cardiovascular processes responsible for disease development in order to develop more effective therapeutic strategies. Recent knowledge gleaned from the study of epigenetic mechanisms in the vasculature has uncovered new potential targets for intervention. Herein, we provide an overview of epigenetic mechanism, and review recent findings related to epigenetics in vascular diseases, highlighting classical epigenetic mechanism such as DNA methylation and histone modification as well as the newly discovered non-coding RNA mechanisms.

The emerging landscape of non-conventional RNA functions in atherosclerosis.

Most of the human genome is transcribed into non-coding RNAs (ncRNAs), which encompass a heterogeneous family of transcripts including microRNAs (miRNAs), long ncRNAs (lncRNAs), circular RNAs (circRNAs), and others. Although the detailed modes of action of some classes are not fully elucidated, the common notion is that ncRNAs contribute to sculpting gene expression of eukaryotic cells at multiple levels. These range from the regulation of chromatin remodeling and transcriptional activity to post-transcriptional regulation of messenger RNA splicing, stability, and decay. Many of these functions ultimately govern the expression of coding and non-coding genes to affect diverse physiological and pathological mechanisms in vascular biology and beyond. As such, different classes of ncRNAs emerged as crucial regulators of vascular integrity as well as active players in the pathophysiology of atherosclerosis from the early stages of endothelial dysfunction to the clinically relevant complications. However, research in recent years revealed unexpected findings such as small ncRNAs being able to biophysically regulate protein function, the glycosylation of ncRNAs to be exposed on the cell surface, the release of ncRNAs in the extracellular space to act as ligands of receptors, and even the ability of non-coding portion of messenger RNAs to mediate structural functions. This evidence expanded the functional repertoire of ncRNAs far beyond gene regulation and highlighted an additional layer of biological control of cell function. In this Review, we will discuss these emerging aspects of ncRNA biology, highlight the implications for the mechanisms of vascular biology and atherosclerosis, and discuss possible translational implications.

rs41291957 controls miR-143 and miR-145 expression and impacts coronary artery disease risk.

The role of single nucleotide polymorphisms (SNPs) in the etiopathogenesis of cardiovascular diseases is well known. The effect of SNPs on disease predisposition has been established not only for protein coding genes but also for genes encoding microRNAs (miRNAs). The miR-143/145 cluster is smooth muscle cell-specific and implicated in the pathogenesis of atherosclerosis. Whether SNPs within the genomic sequence of the miR-143/145 cluster are involved in cardiovascular disease development is not known. We thus searched annotated sequence databases for possible SNPs associated with miR-143/145. We identified one SNP, rs41291957 (G > A), located -91 bp from the mature miR-143 sequence, as the nearest genetic variation to this miRNA cluster, with a minor allele frequency > 10%. In silico and in vitro approaches determined that rs41291957 (A) upregulates miR-143 and miR-145, modulating phenotypic switching of vascular smooth cells towards a differentiated/contractile phenotype. Finally, we analysed association between rs41291957 and CAD in two cohorts of patients, finding that the SNP was a protective factor. In conclusion, our study links a genetic variation to a pathological outcome through involvement of miRNAs.

New Insights on the Emerging Genomic Landscape of CXCR4 in Cancer: A Lesson from WHIM.

Deciphering the molecular alterations leading to disease initiation and progression is currently crucial to identify the most relevant targets for precision therapy in cancer patients. Cancers express a complex chemokine network influencing leucocyte infiltration and angiogenesis. Moreover, malignant cells also express a selective repertoire of chemokine receptors that sustain their growth and spread. At present, different cancer types have been shown to overexpress C-X-C chemokine receptor type 4 (CXCR4) and to respond to its ligand C-X-C motif chemokine 12 (CXCL12). The CXCL12/CXCR4 axis influences cancer biology, promoting survival, proliferation, and angiogenesis, and plays a pivotal role in directing migration of cancer cells to sites of metastases, making it a prognostic marker and a therapeutic target. More recently, mutations in the C-terminus of CXCR4 have been identified in the genomic landscape of patients affected by Waldenstrom's macroglobulinemia, a rare B cell neoplasm. These mutations closely resemble those occurring in Warts, Hypogammaglobulinemia, Immunodeficiency, and Myelokathexis (WHIM) syndrome, an immunodeficiency associated with CXCR4 aberrant expression and activity and with chemotherapy resistance in clinical trials. In this review, we summarize the current knowledge on the relevance of CXCR4 mutations in cancer biology, focusing on its importance as predictors of clinical presentation and response to therapy.

UHRF1 epigenetically orchestrates smooth muscle cell plasticity in arterial disease.

Adult vascular smooth muscle cells (VSMCs) dedifferentiate in response to extracellular cues such as vascular damage and inflammation. Dedifferentiated VSMCs are proliferative, migratory, less contractile, and can contribute to vascular repair as well as to cardiovascular pathologies such as intimal hyperplasia/restenosis in coronary artery and arterial aneurysm. We here demonstrate the role of ubiquitin-like containing PHD and RING finger domains 1 (UHRF1) as an epigenetic master regulator of VSMC plasticity. UHRF1 expression correlated with the development of vascular pathologies associated with modulation of noncoding RNAs, such as microRNAs. miR-145 - pivotal in regulating VSMC plasticity, which is reduced in vascular diseases - was found to control Uhrf1 mRNA translation. In turn, UHRF1 triggered VSMC proliferation, directly repressing promoters of cell-cycle inhibitor genes (including p21 and p27) and key prodifferentiation genes via the methylation of DNA and histones. Local vascular viral delivery of Uhrf1 shRNAs or Uhrf1 VSMC-specific deletion prevented intimal hyperplasia in mouse carotid artery and decreased vessel damage in a mouse model of aortic aneurysm. Our study demonstrates the fundamental role of Uhrf1 in regulating VSMC phenotype by promoting proliferation and dedifferentiation. UHRF1 targeting may hold therapeutic potential in vascular pathologies.

MicroRNA-26a/cyclin-dependent kinase 5 axis controls proliferation, apoptosis and in vivo tumor growth of diffuse large B-cell lymphoma cell lines.

Diffuse large B-cell lymphoma (DLBCL) is the most frequent type of non-Hodgkin lymphoma. Despite a favorable therapeutic response to first-line chemo-immunotherapy, still 30-40% of patients is refractory, or relapse after this treatment. Thus, alternative strategies must be sought. Previous studies have indicated that cyclin-dependent kinase 5 (CDK5), a serine/threonine protein kinase, is involved in tumor development and progression, and it may represent a potential therapeutic target. However, its role in modulating DLBCL growth and progression remains largely unexplored. In this study, we show that CDK5 and its activator, cyclin-dependent kinase 5 activator 1 (CDK5R1 or p35), are overexpressed in DLBCL cell lines and that signal transducer and activator of transcription 3 (STAT3) phosphorylation and activity is dependent on CDK5 expression in DLBCL. Using public data sets, we also demonstrate that patients with DLBCL show a higher expression of CDK5 compared with healthy individuals. By using loss-of-function approaches, we demonstrate that CDK5's activity regulates proliferation and survival of DLBCL cells. MicroRNAs (miRNAs or miRs) are small noncoding RNAs that negatively regulating gene expression and are involved in cancer initiation and progression. We identify miR-26a as direct regulator of p35 expression and CDK5 activity. We show that miR-26a expression is lower in DLBCL cell lines compared to B lymphocytes and that its ectopic expression leads to a drastic reduction of DLBCL tumor growth in vivo and decreased proliferation, cell-cycle progression, and survival in vitro. Remarkably, concomitant overexpression of a 3'-UTR-truncated form of p35 promoted tumor growth in vivo and cell proliferation, cell-cycle progression, and cell survival in vitro. In conclusion, these results demonstrate an important role for miR-26a and CDK5 together in the survival and growth of DLBCL cells, suggesting the existence of potential novel therapeutic targets for the treatment of DLBCL.

MiR-320a as a Potential Novel Circulating Biomarker of Arrhythmogenic CardioMyopathy.

Diagnosis of Arrhythmogenic CardioMyopathy (ACM) is challenging and often late after disease onset. No circulating biomarkers are available to date. Given their involvement in several cardiovascular diseases, plasma microRNAs warranted investigation as potential non-invasive diagnostic tools in ACM. We sought to identify circulating microRNAs differentially expressed in ACM with respect to Healthy Controls (HC) and Idiopathic Ventricular Tachycardia patients (IVT), often in differential diagnosis. ACM and HC subjects were screened for plasmatic expression of 377 microRNAs and validation was performed in 36 ACM, 53 HC, 21 IVT. Variable importance in data partition was estimated through Random Forest analysis and accuracy by Receiver Operating Curves. Plasmatic miR-320a showed 0.53 ± 0.04 fold expression difference in ACM vs. HC (p < 0.01). A similar trend was observed when comparing ACM (n = 13) and HC (n = 17) with athletic lifestyle, a ACM precipitating factor. Importantly, ACM patients miR-320a showed 0.78 ± 0.05 fold expression change vs. IVT (p = 0.03). When compared to non-invasive ACM diagnostic parameters, miR-320a ranked highly in discriminating ACM vs. IVT and it increased their accuracy. Finally, miR-320a expression did not correlate with ACM severity. Our data suggest that miR-320a may be considered a novel potential biomarker of ACM, specifically useful in ACM vs. IVT differentiation.